Liver disease induced by hepatitis C virus (HCV) and hepatitis B virus (HBV) is a global health problem. The World Health Organization estimates that 350-400 million people are chronically infected, and about one million die annually due to chronic hepatitis, cirrhosis or liver cancer. Another 200 million people are infected by HCV, of whom 70%-85% will become chronically infected. HBV and HCV infection is the leading cause of liver disease in Asia. In Western countries, HCV infection is the leading indication for liver transplantation and a major cause of liver cancer.
The liver is a unique organ for immune responses and viruses/tumors (for review, see Crispe, I. N. 2003. Hepatic T cells and liver tolerance. Nat Rev Immunol 3:51-62). The liver intrinsically dampens the immune responses to foreign antigens filtering through it from the intestines. An allogeneic liver transplant is often accepted with minimal or no immune suppression. Tumors with tumor-specific antigens can metastasize to and survive in the liver in immuno-competent patients. Infection of hepatocytes by viruses such as HCV often leads to specific immune tolerance to the virus, and to chronic or persistent infection (Bowen et al. 2005. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature 436:946-52; Grakoui et al. 2003. HCV persistence and immune evasion in the absence of memory T cell help. Science 302:659-62).
Although the liver may provide an immunologically privileged site for infections, most infections in the liver, such as HAV and MHV, and HBV in adults, are effectively cleared and accompanied with lasting protective immunity. It is unusual that greater than 80% of HCV infection in immuno-competent hosts leads to persistent infection. HCV-encoded factors and/or unique host cell tropism may contribute to the efficient immune evasion and HCV persistence.
The liver consists of unique subsets of antigen presenting cells and lymphocytes. In addition to dendritic cells, a large number of liver macrophages (or Kupffer cells) and sinusoidal endothelial cells in the liver also have efficient phagocytosis activity and express various levels of MHC and T cell costimulatory molecules. However, they often show suboptimal T cell activation activity in vivo (Everett et al. 2003. Kupffer cells: another player in liver tolerance induction. Liver Transpl 9:498-9; Parker et al. 2005. Liver immunobiology. Toxicol Pathol 33:52-62; Racanelli et al. 2006. The liver as an immunological organ. Hepatology 43:S54-S62; Sun et al. 2003. Hepatic allograft-derived Kupffer cells regulate T cell response in rats. Liver Transpl 9:489-97; Wiegard et al. 2005. Murine liver antigen presenting cells control suppressor activity of CD4+ CD25+ regulatory T cells. Hepatology 42:193-9). The liver also contains lymphoid cells with unique features. Up to 25% of lymphoid cells belong to the NKT cell population that expresses TCR as well as NK markers. Their function in the liver is not clear, but they have been implicated in clearing infections in the liver (Behar et al. 1999. Susceptibility of mice deficient in CD1 D or TAP1 to infection with Mycobacterium tuberculosis. J Exp Med 189:1973-80; Skold et al. 2003. Role of CD1d-restricted NKT cells in microbial immunity. Infect Immun 71:5447-55).
HCV/HBV coinfection with the HIV-1 virus, which is highly prevalent among intravenous drug users, leads to accelerated liver disease progression (Bani-Sadr et al. 2006. Hepatic steatosis in HIV-HCV coinfected patients: analysis of risk factors. Aids 20:525-31; Brau, N. 2003. Update on chronic hepatitis C in HIV/HCV-coinfected patients: viral interactions and therapy. Aids 17:2279-90; Sabin et al. 2004. HIV/HCV coinfection, HAART, and liver-related mortality. Lancet 364:757-8; author reply 758). Liver failure is increasingly affecting HIV-1/HCV-coinfected patients, as their AIDS-free survival is being prolonged by highly active antiretroviral therapy (HAART).
The available treatment for HCV infection is far from optimal, and HIV-1/HCV-coinfected patients show even worse responses to pegylated interferon plus rivabirin than HCV-monoinfected patients (Sola et al. 2006. Poor response to hepatitis C virus (HCV) therapy in HIV- and HCV-coinfected patients is not due to lower adherence to treatment. AIDS Res Hum Retroviruses 22:393-400). There is a great need for alternative treatment options for hepatitis infection.
A relevant small animal model for research on HCV/HBV infection and pathogenesis is therefore needed. However, HCV fails to infect murine cells due to blocks at multiple steps of the HCV life cycle. HCV and HBV can only infect, establish chronic infection and to lead to liver pathogenesis in humans. Only a reduced chronic infection and immuno-pathogenesis are observed in chimpanzees, which provides the only current non-human animal model for HCV infection (Pietschmann et al. 2003. Tissue culture and animal models for hepatitis C virus. Clin Liver Dis 7:23-43).
The Alb-uPA transgenic mouse, developed in 1990 by Heckel et al. (1990. Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator. Cell 62:447-56) to study plasminogen hyperactivation and therapeutic protocols to prevent bleeding, contains a tandem repeat of four murine uPA genes under the control of an albumin promoter. The transgene overexpression results in profound hypo-fibrinogenemia and accelerated hepatocyte death. Homozygous animals can be rescued by transplantation of murine or human hepatocytes, which undergo rapid proliferation to replace the dying hepatocytes (Mercer et al. 2001. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 7:927-33; Meuleman et al. 2005. Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera. Hepatology 41:847-56; Meuleman et al. 2006. Immune suppression uncovers endogenous cytopathic effects of the hepatitis B virus. J Virol 80:2797-807). Transplanted human hepatocytes can be infected with HBV and HCV (Mercer et al. 2001. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 7:927-33; Meuleman et al. 2006. Immune suppression uncovers endogenous cytopathic effects of the hepatitis B virus. J Virol 80:2797-807).
A molecularly cloned, cell culture-produced hepatitis C virus (HCVcc) genome has been recently shown to support efficient replication in vitro (Blight et al. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972-4; Lindenbach et al. 2005. Complete replication of hepatitis C virus in cell culture. Science 309:623-6) and in vivo (Lindenbach et al. 2006. Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci USA 103:3805-9). The HCVcc is infectious in uPA-SCID mice reconstituted with human hepatocytes, and infection can be serially passaged to a naïve animal.
Infectivity of HCV can be studied in the uPA-SCID mice transplanted with human hepatocytes (Kneteman et al. 2006. Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application. Hepatology 43:1346-53; Lindenbach et al. 2005. Complete replication of hepatitis C virus in cell culture. Science 309:623-6; Mercer et al. 2001. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 7:927-33; Meuleman et al. 2005. Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera. Hepatology 41:847-56). However, immuno-pathogenesis cannot because uPA mice have no immune system. In addition, the uPA-SCID mouse is very sick and not suitable for many studies.
The RagFahγC TKO mouse also allows efficient engraftment of human hepatocytes in a uPA transgene-dependent fashion (Azuma et al. 2007. Robust expansion of human hepatocytes in Fah(−/−)/Rag2(−/−)/I12rg(−/−) mice. Nat Biotechnol 25:903-10). In the B6 Rag/γC DKO background, the fumarylacetoacetate hydrolase (Fah) mutation is crossed to generate the RagFahγC triple KO mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted with human hepatocytes. However, due to lack of a functional immune system (which is not suitable for human immune system development), it is not possible to study HCV/HBV immunopathogenesis in these uPA-SCID/-TKO models.
Thus, a mouse model having both a functional human immune system and a human liver is needed to study HCV/HBV infection, immune responses and pathogenesis.
Two human-mouse chimera models with human lymphoid organs implanted in immunodeficiency mice have been constructed to study HIV-1 infection in vivo. The hu-PBL-SCID mouse is limited due to its lack of human hemato-lymphoid organs and its selective engraftment of xeno-reactive human T cells (Mosier et al. 1988. Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 335:256-9; Mosier et al. 1991. Human immunodeficiency virus infection of human-PBL-SCID mice. Science 251:791-4; Tary-Lehmann et al. 1994. Anti-SCID mouse reactivity shapes the human CD4+ T cell repertoire in hu-PBL-SCID chimeras. J Exp Med 180:1817-27). The SCID-hu Thy/Liv mouse has an intact human thymus organ, which allows investigation of HIV-1 pathogenesis in the thymus (McCune et al. 1991. The SCID-hu mouse: a small animal model for HIV infection and pathogenesis. Annu Rev Immunol 9:399-429; McCune et al. 1988. The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 241:1632-9; Su et al. 1995. HIV-1-induced thymocyte depletion is associated with indirect cytopathogenicity and infection of progenitor cells in vivo. Immunity 2:25-36). However, no human B or myeloid cells and very low levels of human T cells are detected in the peripheral organs or blood. Therefore, no significant primary human immune responses are observed in the model.
A more relevant in vivo non-human animal model that allows hepatitis infection as well as hepatitis and HIV co-infection is, therefore, needed.